A novel alkyne cholesterol to trace cellular cholesterol metabolism and localization

Cholesterol is an important lipid of mammalian cells and plays a fundamental role in many biological processes. Its concentration in the various cellular membranes differs and is tightly regulated. Here, we present a novel alkyne cholesterol analog suitable for tracing both cholesterol metabolism and localization. This probe can be detected by click chemistry employing various reporter azides. Alkyne cholesterol is accepted by cellular enzymes from different biological species (Brevibacterium, yeast, rat, human) and these enzymes include cholesterol oxidases, hydroxylases, and acyl transferases that generate the expected metabolites in in vitro and in vivo assays. Using fluorescence microscopy, we studied the distribution of cholesterol at subcellular resolution, detecting the lipid in the Golgi and at the plasma membrane, but also in the endoplasmic reticulum and mitochondria. In summary, alkyne cholesterol represents a versatile, sensitive, and easy-to-use tool for tracking cellular cholesterol metabolism and localization as it allows for manifold detection methods including mass spectrometry, thin-layer chromatography/fluorography, and fluorescence microscopy.     

Complexin synchronizes primed vesicle exocytosis and regulates fusion pore dynamics

ComplexinII (CpxII) and SynaptotagminI (SytI) have been implicated in regulating the function of SNARE proteins in exocytosis, but their precise mode of action and potential interplay have remained unknown. In this paper, we show that CpxII increases Ca²⁺-triggered vesicle exocytosis and accelerates its secretory rates, providing two independent, but synergistic, functions to enhance synchronous secretion. Specifically, we demonstrate that the C-terminal domain of CpxII increases the pool of primed vesicles by hindering premature exocytosis at submicromolar Ca²⁺ concentrations, whereas the N-terminal domain shortens the secretory delay and accelerates the kinetics of Ca²⁺-triggered exocytosis by increasing the Ca²⁺ affinity of synchronous secretion. With its C terminus, CpxII attenuates fluctuations of the early fusion pore and slows its expansion but is functionally antagonized by SytI, enabling rapid transmitter discharge from single vesicles. Thus, our results illustrate how key features of CpxII, SytI, and their interplay transform the constitutively active SNARE-mediated fusion mechanism into a highly synchronized, Ca²⁺-triggered release apparatus.     

Loss of fused in sarcoma (FUS) promotes pathological Tau splicing

A subset of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) patients present pathological redistribution and aggregation of the nuclear protein fused in sarcoma (FUS) in the cytoplasm. Although FUS associates with the spliceosomal complex, no endogenous neuronal splicing targets have been identified. Here we identify Tau mRNA as a physiological splicing target of FUS. In mouse brain, FUS directly binds to Tau pre-mRNA, and knockdown of FUS in hippocampal neurons leads to preferential inclusion of Tau exons 3 and 10. FUS knockdown causes significant growth cone enlargement and disorganization reminiscent of Tau loss of function. These findings suggest that disturbed cytoskeletal function and enhanced expression of the neurodegeneration-associated Tau exon 10 might contribute to FTLD/ALS with FUS inclusions.     

Mnl2, a novel component of the ER associated protein degradation pathway

In eukaryotes, membrane and soluble proteins of the secretory pathway enter the endoplasmic reticulum (ER) after synthesis in an unfolded state. Directly after entry, most proteins are modified with glycans at suitable glycosylation sites and start to fold. A protein that cannot fold properly will be degraded in a process called ER associated degradation (ERAD). Failures in ERAD, either by loss of function or by premature degradation of proteins, are a cause of severe diseases. Therefore, the search for novel ERAD components to gain better insight in this process is of high importance. Carbohydrate trimming is a relevant process in ER quality control. In this work a novel putative yeast mannosidase encoded by the open reading frame YLR057W was identified and named Mnl2. Deletion of MNL2 diminished the degradation efficiency of misfolded CPY^* in the absence of the cognate mannosidase Mnl1, indicating a specific role in ERAD.     

Markers of enhanced cholesterol absorption are a strong predictor for cardiovascular diseases in patients without diabetes mellitus

Objective

Hypercholesterolemia is a major risk factor for cardiovascular disease (CVD), and diabetes mellitus and statin treatment affect cholesterol metabolism. The aim of the present study was to evaluate markers of cholesterol metabolism and determine their relationship with CVD in patients without diabetes mellitus who were not receiving statin treatment.

Methods

In addition to conventional CVD risk factors, plasma levels of campesterol and sitosterol (indicators of cholesterol absorption) and lathosterol (an indicator of cholesterol synthesis) were determined in 835 consecutive patients referred for coronary angiography. Coronary artery disease was evaluated by coronary angiograms, carotid atherosclerosis and peripheral vascular disease were assessed by Doppler ultrasound, and cerebrovascular accidents and transient ischemic attacks were identified by medical history.

Results

After excluding patients with known diabetes mellitus and those receiving statin treatment, 177 patients were included in the analysis. Compared to patients without CVDs (n = 111), patients with concomitant CVDs (n = 66) had a reduced lathosterol-to-cholesterol ratio (1.25 ± 0.61 vs. 1.38 ± 0.63, P < 0.05) and an increased campesterol-to-cholesterol ratio (1.81 ± 1.04 vs. 1.50 ± 0.69, P < 0.05), indicating that enhanced absorption and reduced synthesis of cholesterol is associated with CVD development. Logistic regression analysis including all established cardiovascular risk factors (age, sex, total cholesterol, arterial hypertension, body mass index and smoking) revealed that campesterol and the campesterol-to-cholesterol ratio were significant predictors of concomitant CVD in this patient population.

Conclusion

In patients without diabetes mellitus, markers of enhanced cholesterol absorption were a strong predictor for concomitant CVD.     

Autocrine embryotropins revisited: how do embryos communicate with each other in vitro when cultured in groups?

In the absence of the maternal genital tract, preimplantation embryos can develop in vitro in culture medium where all communication with the oviduct or uterus is absent. In several mammalian species, it has been observed that embryos cultured in groups thrive better than those cultured singly. Here we argue that group‐cultured embryos are able to promote their own development in vitro by the production of autocrine embryotropins that putatively serve as a communication tool. The concept of effective communication implies an origin, a signalling agent, and finally a recipient that is able to decode the message. We illustrate this concept by demonstrating that preimplantation embryos are able to secrete autocrine factors in several ways, including active secretion, passive outflow, or as messengers bound to a molecular vehicle or transported within extracellular vesicles. Likewise, we broaden the traditional view that inter‐embryo communication is dictated mainly by growth factors, by discussing a wide range of other biochemical messengers including proteins, lipids, neurotransmitters, saccharides, and microRNAs, all of which can be exchanged among embryos cultured in a group. Finally, we describe how different classes of messenger molecules are decoded by the embryo and influence embryo development by triggering different pathways. When autocrine embryotropins such as insulin‐like growth factor‐I (IGF‐I) or platelet activating factor (PAF) bind to their appropriate receptor, the phosphatidylinositol‐4,5‐bisphosphate 3‐kinase (PI3K) pathway will be activated which is important for embryo survival. On the other hand, the mitogen‐activated protein kinase (MAPK) pathway is activated when compounds such as hyaluronic acid and serotonin bind to their respective receptors, thereby acting as growth factors. By activating the peroxisome‐proliferator‐activated receptor family (PPAR) pathway, lipophilic autocrine factors such as prostaglandins or fatty acids have both survival and anti‐apoptotic functions. In conclusion, considering different types of messenger molecules simultaneously will be crucial to understanding more comprehensively how embryos communicate with each other in group‐culture systems. This approach will assist in the development of novel media for single‐embryo culture.     

Der Einfluß der Umweltfaktoren auf das Wachstum und den Häutungsrhythmus der StrandkrabbeCarcinides maenas

Zusammenfassung 1. Junge Strandkrabben von 4–16 mm Carapaxbreite wurden bis zur Geschlechtsreife unter konstanten Umweltbedingungen aufgezogen.2. Die Dauer ihrer Häutungsintervalle nimmt bei konstanter Temperatur mit der Körpergröße stetig zu.3. Die Dauer der Häutungsintervalle hängt von der Temperatur und der Ernährung ab. Von der Tageslänge scheint sie weitgehend unabhängig zu sein.4. Der relative Grösßenzuwachs bei jeder Häutung ist im gesamten untersuchten Größenbereich und bei den verschiedenen Temperaturen bei allen Häutungen gleich: Bei den Häutungen verdoppelt sich jeweils das Körpervolumen.5. Augenstielamputationen und Verlust von Extremitäten wirken auf den Häutungsrhythmus in gleicher Weise: Die Schwankungsbreite in der Dauer der Häutungsintervalle ist vermindert. Die Häutungsintervalle sind in 20° C deutlich, in 10° C nur geringfügig verkürzt.6. Durch die Anwesenheit größerer Artgenossen werden die Häutungen verzögert. Die optische Wahrnehmung spielt dabei keine Rolle.7. Aus diesen Ergebnissen wird folgendes geschlossen: Der ausschlaggebende Faktor für die Auslösung von Häutungen ist ein bestimmter Größenzuwachs. Temperatur und Ernährung beeinflussen den Häutungsrhythmus dadurch, daß sie das Tempo des Wachstums bestimmen. Die winterliche Häutungsruhe in Freilandpopulationen wird nicht durch den Kurztag bedingt, sondern durch die Kälte. Diese hemmt lediglich das Wachstum, sie verhindert nicht die Häutungen über das häutungshemmende Hormon. Dieses vermindert vielmehr die Temperaturabhängigkeit des Häutungsrhythmus, indem es die Häutungen im Warmen stärker verzögert als im Kalten. Es gestattet die Anpassung des Häutungstermins an die individuelle Lage der Tiere. Es hemmt in Anwesenheit größerer Artgenossen die Häutung. Beim Verlust mehrerer Gliedmaßen wird seine Sekretion eingestellt, so daß die nächste Häutung vorzeitig erfolgt. Das häutungshemmende Hormon bedingt dementsprechend die große individuelle Variation in der Dauer der Häutungsintervalle.

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The effect of environmental factors on growth and moulting rhythm in the shore crab,Carcinides maenas

Young crabs (carapace width 4 to 16 mm) were raised under controlled conditions in the laboratory. The time intervals between subsequent moults increase at all test temperatures with increasing body size. The length of intermoult periods varies with temperature and feeding. It is not affected by day length. Moulting takes place as soon as a certain increase in size is attained. In comparable size groups, the amount of this increase is identical in all test temperatures. Moreover, the relation of increase to initial size is constant over the whole size range investigated. The body volume doubles at each moult. Eyestalk amputations and loss of extremities have similar effects: They shorten the intermoult periods at 20° C considerably, but at 10° C they do so only slightly; furthermore, the amplitude of fluctuations is narrowed. The presence of large specimens tends to retard moulting in smaller ones; this response is independent of visual stimuli. The following assumptions are made: Low temperatures retard the moulting rhythm directly by slowing down growth. They are not acting via the moult inhibiting hormone. Loss of several extremities causes a stop of hormone delivery resulting in shortened intermoult periods. Recognition by touch of a larger specimen causes increased hormone delivery and thus retardation of the subsequent moulting process.